COMBINATION OF A SOLUBLE COMPLEMENT RECEPTOR -1(SCR1) AND AN AMIDINOPHENYL OR AMIDINO NAPHTHYL-ESTER FOR TREATING INFLAMMATION

Abstract: A method of treating a disease or disorder associated with inflammation or inappropriate complement activation which method comprises administering to a mammal in need thereof an effective amount of a soluble CR1 protein and an effective amount of an amidinophenyl or amidinonaphthyl ester, including pharmaceutically acceptable salts thereof.

Description: COMBINATION OF A SOLUBLE COMPLEMENT RECEPT0R-1( 5CR1 ) AND AN AMIDINOPHENYL OR AMIDINONAPHTYL ESTER FOR TREATING INFLAMMATION The present invention relates to therapeutic compositions of protease inhibitors and human soluble complement receptor 1 which act synergistically to inhibit activation of complement. Such compositions are useful in the therapy of inflammatory or immune disorders involving complement activation. Complement receptor type 1 (CRl) is present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CRl binds to the complement components C3b and C4b and has also been referred to as the C3b/C4b receptor. The structural organisation and primary sequence of one allotype of CRl is known (Klickstein et ai, 1987, J. Exp. Med. 165:1095-1112, Klickstein et al., 1988, J. Exp. Med. 168:1699-1717; Hourcade er α/.,1988, J. Exp. Med. 168:1255-1270, WO 89/09220, WO 91/05047). It is composed of 30 short consensus repeats (SCRs) that each contain around 60-70 amino acids. In each SCR, around 29 of the average 65 amino acids are conserved. Each SCR has been proposed to form a three dimensional triple loop structure through disulphide linkages with the third and first and the fourth and second half-cystines in disulphide bonds. CRl is further arranged as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a leader sequence, the CRl molecule consists of the N-terminal LHR-A, the next two repeats, LHR-B and LHR- C, and the most C-terminal LHR-D followed by 2 additional SCRs, a 25 residue putative transmembrane region and a 43 residue cytoplasmic tail. Several soluble fragments of CRl have been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNAs being expressed (WO 89/09220, WO 91/05047). The soluble CRl fragments were functionally active, bound C3b and/or C4b and demonstrated Factor I cofactor activity depending upon the regions they contained. Such constructs inhibited in vitro complement- related functions such as neutrophil oxidative burst, complement mediated haemolysis, and C3a and C5a production. A particular soluble construct, sCRl/pBSCRlc, also demonstrated in vivo activity in a reversed passive Arthus reaction (WO 89/09220, WO 91/05047; Yeh et al., 1991, J. Immunol. 146:250- 256), suppressed post-ischemic myocardial inflammation and necrosis (WO 89/09220, WO 91/05047; Weisman et al., Science, 1990, 249:146-151; Dupe, R. et al. Thrombosis & Haemostasis (1991) 65(6) 695.) and extended survival rates following transplantation (Pruitt & Bollinger, 1991, J. Surg. Res 50:350; Pruitt et al., 1991 Transplantation 52; 868), as well as demonstrating therapeutic inhibition of complement activation in several animal models of disease such as lung injury (Rabinovici et al, 1992 J. Immunol. 149:1744-1750; Mulligan et al, 1992 J. Immunol. 148:3086-3092), intestinal ischaemia (Hill et al, 1992 FASEB J. 6:A1049) and acute myocardial infarction (Weisman et al, 1990 Science 249:146-151, Dupe et α/,1991 (above)). In a number of cases, the doses of sCRl required for therapeutic effects in these models were large (>5mg/kg). Because sCRl is a biopharmaceutical produced by mammalian cell culture techniques, it is desirable to reduce the dose and hence the cost of therapy. Certain amidinophenyl and amidinonaphthyl esters of carboxylic acids are known to be inhibitors of complement activation as well as having antitrypsin, antiplasmin, antikallikrein and antithrombin activity (GB 2095-239, GB 2083-818). GB 2083818 discloses compounds of formula (A): wherein Z represents -(CH2) a -, -(CH2)b"CH(R3)-, -CH=C(R4)- or -O-CH(R4)-, where a is 0, 1, 2 or 3, b is 0, 1 or 2, R3 is a straight or branched chain alkyl group of 1 to 4 carbon atoms or a cycloalkyl group of 3 to 6 carbon atoms, and R4 is a hydrogen atom or a straight or branched chain alkyl group of 1 to 4 carbon atoms and wherein the -CH(R3)-, = C(R4)- or -CH(R4)- moiety is bonded to the -COO group; and R and R2, which may be the same or different, represent each a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, -O-R5, -S-R5, -COOR5, -COR6, -O-COR7, -NHCOR7, -(CH 2 ) c -NRgR 9 , -SO2NRgRo, NO2, CN, halogen, CF3, methylenedioxy, where c is 0, 1 or 2; R5 is a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, or benzyl group; Rg is a hydrogen atom or straight or branched chain alkyl group of 1 to 4 atoms; R7 is a straight or branched chain alkyl group of 1 to 4 carbon atoms; Rg and R9, which may be the same or different, are each a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, or amino radical protecting group; and RI Q is a hydrogen atom, dimethyl or CF3. GB 2095239 discloses compounds of the general formula (B): wherein Ri 1 represents a straight or branched chain alkyl group 1 to 6 carbon atoms, a straight or branched chain alkenyl group of 2 to 6 carbon atoms having 1 to 3 double bonds, Rl3-(CH 2 ) d -, Ri4-(CH 2 )e-, where R 13 is a cycloalkyl group of 3 to 6 carbon atoms or a cycloalkenyl group of 3 to 6 atoms having 1 or 2 double bonds; d is 0, 1, 2 or 3; R 14 is an amino or guanidino group or a protected amino or guanidino group; e is a number from 1 to 5; R15 and Rjg, which may be the same or different, are each a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms, -OR 17, methylenedioxy group, -SR17, -COOR17, -CORig, -OCOR^, -NHCOR19, -(CH 2 )f-NR 2 oR21 ( f is 0, 1,2), -SO 2 NR oR2b a halogen atom, -CF3, NO2, CN, Rl7 is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms or a benzyl group; Ri is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms; Ri 9 is a straight or branched alkyl group of 1 to 4 carbon atoms; R20 and R21, which may be the same or different, are each a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms, or an amino-protecting group; R22 is O, S or NH; R23 is a 2',3 '-dimethyl or 3'-CF 3 group; Y is -(CH 2 ) g -(g is 0, 1, 2 or 3), -(CH 2 )h-CHR 2 4- (h is 0, 1 or 2), or -CH=CR 2 5S R24 is a straight or branched alkyl group of 1 to 4 carbon atoms and the carbon atom or the CHR24 moiety is attached to the COO group; R25 is a hydrogen atom or a straight or branched alkyl group of 1 to 4 carbon atoms and the carbon atom of the CR25 moiety is attached to the COO group; and R12 represents -R26> ~ OR26. -COOR27, one or two of the same halogen atoms, -NH2' -SO3H, wherein R26 is a straight or branched alkyl group of 1 to 4 carbon atoms; R27 is a hydrogen atom or a straight or branched alkyl group of 1 to 4 carbon atoms; and R2g is a hydrogen atom or a guanidino group. Other amidinophenyl esters of carboxylic acids are also known to inhibit proteases of the coagulation pathway (A.D.Turner et al, 1986 Biochem. 25:4929-35) and have also been employed to acylate the active centres of fibrinolytic enzymes reversibly (US 4,285,932, US 4,507,283, EP 0,297,882, R.A.G.Smith et al, 1985 Progress in Fibrinolysis VII 227-231). US 4285932, US 4507283 and EP 0297882 disclose compounds of formula (C): wherein R x is benzoyl optionally substituted with one or two substituents independently selected from halogen, Ci .g alkyl, C2-6 alkenyl, C\.* alkoxy, C\. alkanoyloxy, C\.β alkanoylamino, amino, dimethylamino or guanidino; naphthoyl; or acryloyl optionally substituted with Ci _g alkyl, furyl or phenyl wherein the phenyl moiety is optionally substituted with Cj.g alkyl. Thus this type of compound is not a specific inhibitor of the proteases of the complement system. Synergistic compositions of CRl -related polypeptides with certain organic compounds have been described (W092/10205). According to the present invention there is provided a method of treating a disease or disorder associated with inflammation or inappropriate complement activation which method comprises administering to a mammal in need thereof an effective amount of a soluble CRl protein and an effective amount of an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity: HN O >~ ® - OC-(B) (I) H 2 N wherein A is phenyl optionally substituted with Cj_4 alkyl, Cj_4 alkoxy, C1.4 alkoxycarbonyl, halo, NH2 > sulphonyl, benzoyl or C1.4 alkylbenzoylamino or naphthyl; and B is CH2=CH- optionally substituted by a group selected from C\. alkyl, phenyl and phenyl substituted with Ci.g alkyl; phenyl optionally substituted with one or two substituents independently selected from halogen, C1. alkyl, C2- alkenyl, Cj.g alkoxy, C1 _g alkenoyloxy, Cj.g alkanoylamino, amino, dimethylamino or guanidino; or naphthyl, including pharmaceutically acceptable salts thereof. Preferably, A is phenyl optionally substituted in the 2- or 3- position by halogen and the amidine substituent is in the 4-position of the phenyl ring. B is preferably phenyl 4- substituted by C 1.4 alkoxy and optionally further substituted by halogen. Most preferably, B is 4-methoxyphenyl and A is phenyl or 2-bromophenyl, 4-substituted by the amidine group. Suitable examples of halo include chloro and bromo. Pharmaceutically acceptable salts may be formed with pharmaceutically acceptable acids, for example, maleic, hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, mandelic, tartaric, methanesulphonic and oxalic acid. In a preferred aspect, the soluble CRl component used in combination therapy is encoded by a nucleic acid vector selected from the group consisting of pBSCRlc, pBSCRls, pBM-CRlc, pBSCRlc/pTCSgpt and pBSCRls/pTCSgpt, and is especially that obtainable from pBSCRlc/pTCSgpt, as described in WO 89/09220. The amounts of each compound are chosen such that the concentration of each component required to inhibit by 50% haemolysis of sensitized erythrocytes in a standard complement assay is lowered compared with that required for the individual components in the same assay. This increase in potency is described by a synergy factor which is defined in more detail below. The invention also provides the use of a soluble CRl protein and an amidinophenyl or amidinonaphthyl ester having complement inhibitory activity in the manufacture of a medicament for the treatment of a disease or disorder associated with inflammation or inappropriate complement activation. The compounds may be administered by standard routes, such as, for example, intravenous infusion or bolus injection, and may be administered together or sequentially, in any order. When the compounds are administered together they are preferably given in the form of a pharmaceutical composition comprising both agents. Thus, in a further aspect of the invention there is provided a pharmaceutical composition comprising a soluble CRl protein and an amidinophenyl or amidinonaphthyl ester having complement inhibitory activity together with a pharmaceutically acceptable carrier. In a preferred embodiment, the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. In a further aspect, the invention therefor provides a method for the preparation of a pharmaceutical composition of the invention, which method comprises admixing the combination of soluble CRl protein and an amidinophenyl or amidinonaphthyl ester of formula (I), including pharmaceutically acceptable salts thereof. The present invention also provides a method of treating a disease or disorder associated with inflammation or inappropriate complement activation comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition of the invention. In the above methods, the subject is preferably a human. An effective amount of the protein for the treatment of a disease or disorder is in the dose range of 0.01-lOOmg/kg; preferably 0.1-lOmg kg. An effective amount of the ester for the treatment of a disease or disorder is in the dose range 0.05-100 mg/kg; preferably 0.05-10 mg/kg. The ratio of protein to ester is preferably in the range 1:1 to 1:20 by weight. The composition typically contains a therapeutically active amount of the protein and ester and a pharmaceutically acceptable excipient or carrier such as saline, buffered saline, dextrose, or water. Compositions may also comprise specific stabilising agents such as sugars, including mannose and mannitol, and local anaesthetics for injectable compositions, including, for example, lidocaine. A pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition is also within the scope of the invention. The present invention also provides a method for treating a thrombotic condition, in particular acute myocardial infarction, in a human or non-human animal, said method comprising administering to the patient a composition according to this invention. This invention further provides a method for treating adult respiratory distress syndrome (ARDS) in a human or non-human animal, said method comprises administering to the patient a composition according to this invention. The invention also provides a method of delaying hyperacute allograft or hyperacute xenograft rejection in a human or non-human animal which receives a transplant by administering a composition according to this invention. The methods and compositions of this invention are useful in the treatment of complement-mediated or complement-related disorders, including but not limited to those listed below. Disease and Disorders Involving Complement Neurological Disorders multiple sclerosis stroke Guillain Barre Syndrome traumatic brain injury Parkinson's disease allergic encephalitis Disorders of Inappropriate or Undesirable Complement Activation hemodialysis complications hyperacute allograft rejection corneal graft rejection xenograft rejection interleukin-2 induced toxicity during IL-2 therapy paroxysmal nocturnal haemoglobinuria Inflammatory Disorders inflammation of autoimmune diseases Crohn's Disease adult respiratory distress syndrome thermal injury including burns or frostbite uveitis Post-Ischemic Reperfusion Conditions myocardial infarction balloon angioplasty post-pump syndrome in cardiopulmonary bypass or renal hemodialysis renal ischemia hepatic ischemia Infectious Diseases or Sepsis multiple organ failure septic shock Immune Complex Disorders and Autoimmune Diseases rheumatoid arthritis systemic lupus erythematosus (SLE) SLE nephritis proliferative nephritis glomerulonephritis hemolytic anemia myasthenia gravis Reproductive Disorders antibody- or complement-mediated infertility MATERIALS BRL 55730 - is the soluble complement receptor type 1 derived from the expression of plasmid pBSCRlc/pTCSgpt in CHO cells (WO 89/09220). BRL24894A (APAN) - 4-amidinophenyl 4'-methoxybenzoate HC1 (EP-0009879) BRAPAN - 4-amidino-2-bromophenyl 4'-methoxybenzoate HC1 (Example 3). METHODS Anti-complement Activity Measured by the Haemolysis of Sheep Erythrocytes Functional activity of complement inhibitors was assessed by measuring the inhibition of complement mediated lysis of sheep erythrocytes sensitised with rabbit antibodies (obtained from Diamedix Corporation, Miami, USA). Human serum diluted 1:125 or 1/35.7 in 0.1 M Hepes pH 7.4/ 0.15 M NaCl buffer was the source of complement and was prepared from a pool of volunteers essentially as described in Dacie & Lewis, 1975 (Practical Haematology 5th Edition, Churchill Livingstone, Edinburgh and New York, pp3-4). Briefly, blood was warmed to 37°C for 5 minutes, the clot removed and the remaining serum clarified by centrifugation. The serum fraction was split into small aliquots and stored at -196°C. Aliquots were thawed as required and diluted in the Hepes buffer immediately before use. Inhibition of complement-mediated lysis of sensitised sheep erythrocytes was measured using a standard haemolytic assay using a v-bottom microtitre plate format as follows, essentially as described by Weisman et al 1990 (above). Standard assay 25 μl of a range of concentrations of inhibitor (typically in the region of O.lμg/ml - 0.00078μg/ml final for BRL55730 and 100 - O.lμM final of APAN or BRAPAN) diluted in Hepes (0.1M Hepes pH7.4/0.15M NaCl) buffer were incubated with 25 μl of buffer and 50 μl of the 1/125 diluted serum for 15 minutes at 37°C. 100 μl of prewarmed sensitised sheep erythrocytes were added for 1 hour at 37°C in a final reaction volume of 200 μl . Samples were spun at 300g at 4°C for 15 minutes before transferring 150 μl of supernatant to flat bottom microtitre plates and determining the absorption at 410 nm, which reflects the amount of lysis in each test solution. Maximum lysis was determined by incubating serum with erythrocytes in the absence of any inhibitor (E+S) from which the proportion of background lysis had been subtracted (determined by incubating erythrocytes with buffer) (E). The background lysis by inhibitor was assessed by incubating inhibitor with erythrocytes (E+I) and then subtracting that from test samples (E+I+S). Inhibition was expressed as a fraction of the total cell lysis such that IH50 represents the concentration of inhibitor required to give 50% inhibition of lysis. For experiments in which serum had been diluted 1/35.7, the incubation time was reduced to 15 mins at 37°C. Otherwise conditions were the same. Maximum Lysis: A max = (E+S) - (E) Lysis in presence of inhibitor: Ao = (E+I+S) - (E+I) Amount of inhibition: IH = (Amax-Ao) Amax Plots were made of [inhibitor] vs IH and IH50 values were determined from the titration curve by reading off the concentration corresponding to IH=0.5. Synergy Assays The assay was carried out in a similar manner to that described above except that inhibitor 1 eg BRL55730 was titrated in the presence of a fixed concentration of inhibitor 2 eg APAN. This was carried out by adding 25 μl of inhibitor 1 to 25 μl of inhibitor 2 in the presence of serum and measuring the degree of lysis as described above. Determination of the Synergy Factor For each synergy experiment both inhibitors were titrated on their own as well as together. EXAMPLE 1 Inhibitor 1, BRL55730 was titrated on its own and in the presence of various concentrations of inhibitor 2, APAN. A plot was made of the [BRL55730] vs IH with and without APAN (Fig.l). The IH50 of BRL55730 was estimated at each APAN concentration. A second plot of [APAN] vs IH was made (Fig.2) from which the IH corresponding to the concentration of APAN used in the synergy experiment was estimated. The results were then tabulated (Table 1). Column 1 refers to the concentration of APAN. The proportion of inhibition that a particular concentration of APAN contributes was estimated from the plot of [APAN] vs IH (Fig.2) (column 2). The IH50 for BRL55730 was determined at each concentration of APAN (column 3). The contribution that a particular concentration of APAN made to the IH50 of BRL55730 was subtracted ie 0.5 - IH (APAN) (column 4). This value was used to read off the concentration of BRL55730 which alone would have provided this level of inhibition (column 5). The adjusted BRL55730 concentration was divided by the measured IH50 to give the synergy factor i.e. column 5/column 3 = column 6. If the effect of APAN was additive, the synergy factor would be 1; values greater than 1 represent a synergistic effect and the greater the value, the greater the degree of synergy. Isobologram Analysis The EH50's of inhibitor 1, eg BRL55730 and inhibitor 2, eg APAN were determined separately. These experimentally determined values are plotted on d e axes of the isobologram and were connected by a straight line, termed the line of additivity (Fig. 3). This line represents combinations of the two inhibitors which, when used together, would result in 50% inhibition (Tallarida (1992) Pain 49: 93-97, Miaskowski & Levine, (1992) 51: 383-387). Hence points falling on the line of additivity indicate an additive effect, points above this line indicate antagonism and points below this curve indicate synergy. BRL55730 was titrated in the presence of fixed concentrations of APAN and the IH50 of BRL55730 determined at each APAN concentration. This data was plotted on the isobologram (Fig. 3). Statistical Analysis of Synergy using Fixed Concentration Pairs. BRL55730 at concentration x which was below its IH50 was assayed in the standard assay. APAN at concentration y which was below its IH50 was also assayed. Then BRL55730r x ι was assayed together with APANryi in the same haemolysis assay. The amount of inhibition was calculated for the two inhibitors when assayed separately and when assayed together. If synergy occurs then the inhibition of the compounds assayed together should be greater than the sum of the two inhibitors separately ie BRL55730[ x ]/APAN ty ] > BRL55730 [x ] + APAN[ y] The data was analysed statistically by t-test according to the formula listed below. Group Mean Sample Standard Standard error size error of the of the mean mean squared a a n fl s.e.m. a (s.e.m.) a 2 BRL55730 b b "b s.e.m. b (s.e.m)h 2 APAN ab ab "ah s.e.m ab (s.e.m.) ab 2 BRL55730/APAN Null Hypothesis = HO ab = a + b (ie effect is additive) Alternative Hypothesis = Hl ab ≠ a + b (ie effect is not additive) t = ab - a - b Equation 1 {(s.e.m. ab 2 ) + (s.e.m. a 2 ) + (s.e.m b 2 )} t was compared with critical levels in t-tables where degrees of freedom (df) df = + n b + n ab - 3 a. Synergy of APAN with BRL55730 in Serum Diluted 1/125 BRL 24894A (APAN) molecular weight 324.24 was made 50 mM in dimethylsulphoxide (DMSO). BRL55730 (in lOmM sodium phosphate pH7.2 buffer) was at 5.3 mg/ml. Both inhibitors were titrated in the standard assay over the concentration range of 100 μM - 0.78 μM for APAN and 0.125 μg/ml - 0.00098 μg/ml for BRL55730. Two titration curves were performed for BRL55730 from which the mean IH50 was determined as 0.01 μg/ml (Fig.l) and one curve for APAN from which the IH50 was determined as 10 μM (Fig.2). To test for synergy, BRL55730 was titrated over the same concentration range but in the presence of fixed concentrations of APAN from 1 - 6 μM for each titration (Fig.l). From the data the synergy factor was calculated as described above. Table 1: Determination of the Synergy Factor for BRL55730 and APAN. From the data in Table 1, inclusion of 6 μM APAN with BRL55730 reduces the IH50 by approximately 8 fold. The calculated synergy factor at each concentration of APAN is given in Table 1 and shows that the effect of APAN is more than additive since the synergy factor is > 1. The synergy factor also remains fairly constant across the range of concentrations used with a mean value of 1.8. BRL55730, concentration range 0.04 - 0.000039 μg/ml, was titrated on its own; the concentration at which no inhibition of complement activation occurred was found to be ~ 0.0004 μg/ml. Titration of APAN on its own showed that a concentration of 4 μM gave an IH of 0.31 and 2 μM gave an IH of 0.16. When BRL55730 was titrated in the presence of APAN at 4 and 2 μM, the inhibition at 0.0004 μg/ml of BRL55730 was greater than that could be accounted for by APAN only showing that APAN potentiates the activity of BRL55730 below the no-effect concentration. b. Isobologram Analysis BRL55730 with APAN The additivity line was constructed as described above taking the data from Figs. 1 & 2. The IH50's of BRL55730 at each APAN concentration (columns 1 & 3 of Table 1 respectively) were then plotted on the isobologram as shown in Fig. 3. The points fall below the line of additivity indicating that the interaction is synergistic. c. Statistical Analysis of Synergy Between BRL55730 and APAN using Fixed Concentration Pairs The following concentration pairs were tested for synergy as described above (i) 0.005 μg/ml BRL55730 4 μM APAN (ii) 0.005 μg/ml BRL55730 2 μM APAN (iii) 0.002 μg/ml BRL55730 4 μM APAN (iv) 0.002 μg/ml BRL55730 2 μM APAN The statistical parameters are given below for each concentration pair. Table 2: Statistical Analysis of Concentration Pair (i) PARAMETER BRL55730 APAN BRL55730 + APAN 0.005 μg/ml 4 μM 0.005 μg/ml + 4 μM MEAN IH 0.312 0.297 0.676 SEM 0.0117 0.0092 0.0096 (SEM) 2 0.000136 0.000086 0.000092 n 14 14 14 df 39 t-value 3.822 At 39 df, probablility of 0.9995 t = 3.558 Calculated t > 3.558. Therefore a + b ≠ ab Table 3: Statistical Analysis of Concentration Pair (ii) PARAMETER BRL55730 APAN BRL55730 + APAN 0.005 μg/ml 2 μM 0.005 μg/ml + 2 μM MEAN IH 0.251 0.124 0.509 SEM 0.0135 0.0183 0.00947 (SEM) 2 0.000183 0.000334 0.0000897 n 16 16 16 df 45 t-value 5.407 At 45 df, probablility of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b ≠ ab Table 4: Statistical Analysis of Concentration Pair (iii) PARAMETER BRL55730 APAN BRL55730 + APAN 0.002 μg/ml 4 μM 0.002 μg/ml + 4 μM MEAN IH 0.123 0.337 0.564 SEM 0.00741 0.00972 0.00691 (SEM) 2 0.000055 0.0000945 0.0000477 n 16 16 16 df 45 t-value 7.451 At 45 df, probablility of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b ≠ ab Table 5: Statistical Analysis of Concentration Pair (iv) PARAMETER BRL55730 APAN BRL55730 + APAN 0.002 μg/ml 2 μM 0.002 μg/ml + 2 μM MEAN IH 0.121 0.192 0.422 SEM 0.0104 0.0106 0.00886 (SEM) 2 0.000109 0.000112 0.0000784 n 16 16 16 df 45 t-value 6.267 At 45 df, probablility of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b ≠ ab At each of the tested concentration pairs, the alternative hypothesis was shown to be correct ie that the effect was not additive and since a + b < ab, synergy has been demonstrated. d. Synergy Effect of BRL55730 on APAN The effect of BRL55730 on the IH50 of APAN was tested in the same way as described in Example la but in this instance APAN was titrated over the range 0.78μM to lOOμM in the presence of fixed concentrations of BRL55730 between 0.001 - 0.006 μg/ml. The effect of BRL55730 on the IH50 of APAN is given in Table 2 and shows that addition of 0.006 μg/ml of BRL55730 shifts the IH50 of APAN from 10 μM to 1 μM which is an improvement of 10 fold. The synergy factor was determined as described in the Methods and found to be > 1 (Table 6) indicating that the synergy process between APAN and BRL55730 is reversible. Unlike the previously described Example la, the synergy factor appears to be dependent on the concentration of BRL55730. Table 6: Determination of the Synergy Factor for APAN and BRL55730 e. Isobologram Analysis of APAN with BRL55730 Isobologram analysis was performed as described above using data from Fig. 2 and Table 6. Data points fell below the line of additivity which indicated the effect was synergistic. f. Synergy of APAN with BRL55730 in Serum Diluted 1/35.7 To test whether the synergy seen between APAN and BRL55730 is reproducible in more concentrated serum, experiments were carried out in serum that had been diluted 1/35.7 which was 3.5 fold more concentrated than described in Example la. As a preliminary test to make sure that this concentration of serum did not produce maximum lysis of the sensitised sheep erythrocytes, 50 μl of serum at various dilutions from 1/10 to 1/150 were preincubated with 50 μl of 0.1 M Hepes pH 7.4 / 0.15 M NaCl buffer at 37°C for 15 mins. 100 μl of erythrocytes were added and samples were incubated for either 15 mins or 30 mins at 37°C. Unlysed cells were spun down at ~ 300 g at 4°C for 15 mins and then 150 μl of supernatant were transfered to a flat bottom microtitre plate before reading the absorbance at 410 nm. A plot of the serum dilution vs absorbance showed that incubation of serum at 1/35.7 dilution for 15 mins with erythrocytes gave ~ 90% of the maximum lysis. Since this concentration of serum proved suitable, synergy experiments of BRL55730 with APAN were carried out in a similar manner as described in Example la except that more concentrated serum was used and incubation times were reduced to 15 mins. Titration of BRL55730 in the more concentrated serum gave an IH50 of ~ 0.14 μg/ml (mean of two determinations) which is 14 fold greater than in 1/125 diluted serum. Similarly the IH50 of APAN was found to be 52 μM (mean of two determinations) which is about five fold greater than in the more dilute serum. The synergy experiments were carried out by titrating BRL55730 over a concentration range of 1 - 0.0078 μg/ml in the presence of APAN at concentrations ranging from 2 - 18 μM. The summary of the data is given in Table 7 and demonstrates that the synergy effect can be extended to more concentrated serum. The synergy factor in this instance appears to be dependent on the concentration of APAN. Table 7: Synergy Value Between BRL55730 and APAN in more concentrated Serum g. Isobologram Analysis of BRL55730 and APAN in More Concentrated Serum The IH50's of BRL55730 and APAN in serum diluted 1/35.7 were used to construct the additivity line. Data points from columns 1 and 3 of Table 7 were used to construct an isobologram. All the points except at 2 μM fall below the addivitity line indicating synergy. The 2 μM data point shows a synergy factor of 1.03 in Table 7 and falls on the line of additivity in the isobologram showing that at this concentration the effect may be additive. h. Synergy Effect of BRAPAN on BRL55730 4-Amidino-2-bromophenyl 4'-methoxybenzoate HC1 (BRAPAN) molecular weight 386 was made 10 mM in DMSO and titrated as described in the Methods using serum diluted 1/125. From two separate determinations the mean value for the EH50 of BRAPAN was 3 μM. A single titration curve of BRL55730 from 0.1 - 0.00078 μg/ml was determined which gave an IH50 of 0.021 μg/ml. To test for synergy BRL55730 was titrated over the same concentration range but in the presence of BRAPAN ranging from 0.1 μM to 0.9μM. Table 8 demonstrates the effect and synergy potential of BRAPAN on BRL55730. As with APAN at the same serum dilution the synergy factor is >1 indicating that BRAPAN synergises with the BRL55730. The synergy factor remains fairly constant over the concentration range giving a mean value of 1.7 which again is very similar to that seen for APAN. Table 8: Synergy Value Between BRL55730 and BRAPAN i. Isobologram Analysis of the effect of BRAPAN on BRL55730 Using the data described in Example lh for BRAPAN, an isobologram was constructed. All the data points fell below the line of additivity indicating synergy was occurring. EXAMPLE 2 Co-formulation of sCRl (BRL 55730) and APAN (BRL 24894A) D-Mannitol (Sigma.UK, 60mg) was dissolved in water for injection (9.5ml). APAN was disssolved in HPLC-grade methanol to a final concentration of 6 mg/ml by stiring at ambient temperature (20-25°C) for 5 min. The solution (0.5ml) was added IMMEDIATELY to the mannitol solution and mixed by shaking. A solution of BRL 55730 (5mg ml in lOmM sodium phosphate pH 7.2, 0.2ml) was added, shaken and immediately frozen in solid CO2- The material was lyophilised at an average pressure of 2-3 mbar and a condenser temperature of -60°C for 20 hours. The white solid had the following composition and was stored desiccated at -70°C: BRL 55730: lmg; BRL 24894A: 3mg; D-Mannitol: 60mg; sodium phosphate: trace. EXAMPLE 3 Preparation of 4-Amidino 2-bromophenyl 4'-methoxybenzoate HCI (BRAPAN) This material was prepared in two steps from 2-bromo 4-cyanophenol. a: Preparation of 2-bromo-4-amidinophenoI hydrochloride 2-Bromo-4-cyanophenol (1.35 g, 6.8 mmole) was dissolved in ethanol (20 ml). Hydrogen chloride gas was passed through the cooled solution, a white precipitate forming after 45 mins. After 2 hours, the bubbling was stopped and die solid in solution placed at 4°C for two days. The white solid was isolated by filtration. This was rapidly suspended in ethanol solution (50 ml) and a saturated solution of ammonia in ethanol (75 ml) was added. The suspension went clear almost immediately and was stirred for 24 hours and allowed to stand for a similar period. All volatile material was removed and the white solid was taken up in water (20 ml). Addition of concentrated hydrochloric acid (5 ml) led to the rapid formation of a white crystalline mass which was isolated by filtration and recrystallised from ethanol (20 ml) and diethyl ether (100 ml). Yield: 860 mg (50%). mp: 279-80°. *H nmr (CDCl 3 -d 6 DMSO) 5: 9.2 (4H, br d, amidine), 8.2 (IH, d, I=2Hz; aryl-H), 7.8 (IH, m, aryl-H), 7.25 (IH, J=9Hz, aryl-H) Infrared (nujol): 3325, 3125, 2300-3400, 1670, 1610, 1585, 1410, 1300, 1175, 1045, 880, 835, 725, 620 cm- 1 Analysis: C 33.45, H 3.28, N 10.95% C 7 HgN 2 OBrCl requires C 33.43, H 3.21, N 11.14% b. Preparation of the title compound To a solution of 4-methoxybenzoyl chloride (271 mg, 1.59 mmole) in dry pyridine (5 ml) was added 2-bromo-4-amidinophenol hydrochloride (400 mg, 1.59 mmole). The initial suspension became a clear solution and then a precipitate reformed. After 1 hour stirring infrared analysis showed the formation of an ester. T e pyridine was removed at reduced pressure the last traces by azeotrope with ethanol. The white solid obtained was recrystallised twice from 5:1 diethyl ether/ethanol (- 100 ml) to leave the white title compound. Yield: 225 mg (37%). mp 212-3°C. l H nmr (d 6 DMSO-trace CDCI3) δ: 9.55 (4H, br, amidinophenol NH), 6.95-8.25 (7H, m, aryl-H), 3.9 (IH, s, OCH3) Infrared (nujol): 2500-3400, 1735, 1665, 1605, 1580,1260, 1230, 1170, 1070, 1020, and 830 cm" 1 Analysis: C 46.66, H 3.74, N 7.19% Ci5,H 12 ,N ,Br,Cl requires C 46.72%, H 3.66%, N 7.26% In the figures: Fig. 1 shows the effect of different concentrations of APAN on BRL 55730; Fig. 2 shows the inhibition of complement activation by APAN; and Fig. 3 is an isobologram of BRL 55730 and APAN in a standard assay.

Claims: CLAIMS 1. A method of treating a disease or disorder associated with inflammation or inappropriate complement activation which method comprises administering to a mammal in need thereof an effective amount of a soluble CRl protein and an effective amount of an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity: HN. ° }-® -OC-® (I) H 2 N wherein A is phenyl optionally substituted with C1.4 alkyl, C1.4 alkoxy, Cι_4 alkoxycarbonyl, halo, NH2, sulphonyl, benzoyl or C 1.4 alkylbenzoylamino or naphthyl; and B is CH2=CH- optionally substituted by a group selected from Cι_6 alkyl, phenyl and phenyl substituted with Cj.g alkyl; phenyl optionally substituted with one or two substituents independently selected from halogen, Ci _g alkyl, C2-6 alkenyl, C1.5 alkoxy, C g alkenoyloxy, Cγ.^ alkanoylamino, amino, dimethylamino or guanidino; or naphthyl, including pharmaceutically acceptable salts thereof. 2. The use of a soluble CRl protein and an amidinophenyl or amidinonaphthyl ester of formula (I) as defined in claim 1 having complement inhibitory activity in die manufacture of a medicament for the treatment of a disease or disorder associated with inflammation or inappropriate complement activation. 3. A pharmaceutical composition comprising a soluble CRl protein and an amidinophenyl or amidinonaphthyl ester of formula (I) as defined in claim 1 having complement inhibitory activity together with a pharmaceutically acceptable carrier. 4. A method of treating a disease or disorder associated with inflammation or inappropriate complement activation comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition of claim 3. 5. A pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of claim 3. 6. A method for the preparation of a pharmaceutical composition according to claim 3, which method comprises admixing the combination of soluble CRl protein and an amidinophenyl or amidinonaphdiyl ester of formula (I) as defined in claim 1. 7. A method, use, composition, method, pack or method according to claim 1, 2, 3, 4, 5 or 6, respectively, wherein the soluble CRl protein is mat encoded by the nucleic acid vector pBSCRlc/pTCSgpt and the ester is 4-amidinophenyl 4'-methoxybenzoate HC1 or 4-amidino-2-bromophenyl 4'-methoxybenzoate HC1.

Inventors: MOSSAKOWSKA DANUTA EWA IRENA, SMITH RICHARD ANTHONY GODWIN, MOSSAKOWSKA, DANUTA, EWA, IRENA, SMITH, RICHARD, ANTHONY, GODWIN
Applicants: MOSSAKOWSKA DANUTA EWA IRENA, SMITH RICHARD ANTHONY GODWIN, SMITHKLINE BEECHAM PLC, MOSSAKOWSKA, DANUTA, EWA, IRENA, SMITH, RICHARD, ANTHONY, GODWIN
Application year: 1994
Main classification: A61K37
IPC codes: A61K31, A61K38, A61P29